Impacts of allergic airway inflammation on lung pathology in a mouse model of influenza A virus

22 Pages·2017·3.19 MB·English

RESEARCHARTICLE
Impacts of allergic airway inflammation on
lung pathology in a mouse model of influenza
A virus infection
AkiraKawaguchi1,2,TadakiSuzuki1*,YukiOhara1,KentaTakahashi1,YukoSato1,
AkiraAinai1,2,NoriyoNagata1,MasatoTashiro2,HidekiHasegawa1
1 DepartmentofPathology,NationalInstituteofInfectiousDiseases,Shinjuku,Tokyo,Japan,2 Centerfor
InfluenzaVirusResearch,NationalInstituteofInfectiousDiseases,Musashimurayama,Tokyo,Japan
*[email protected]
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Abstract
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InfluenzaAvirusistherespiratorypathogenresponsibleforinfluenza.Infectionbythe2009
pandemicinfluenzaA(H1N1)viruscausedseverelowerairwayinflammationandpneumo-
nia.Asthmaisachronicinflammatorydisorderoftheairwaysthataffectstheentirebrachial
tree,andwasoneofthecommonestunderlyingmedicalconditionsamongpatientshospital-
OPENACCESS
izedwiththe2009pandemicinfluenzavirusinfection.Althoughrespiratoryvirusinfections
Citation:KawaguchiA,SuzukiT,OharaY,
arethemajorcausesofasthmaexacerbation,themechanismbywhichinfluenzaexacer-
TakahashiK,SatoY,AinaiA,etal.(2017)Impacts
batesasthmaispoorlyunderstood.Animalmodelsofdiseasecomorbidityarecrucialto
ofallergicairwayinflammationonlungpathology
inamousemodelofinfluenzaAvirusinfection. understandinghost-pathogeninteractionsandelucidatingcomplexpathologies.Existing
PLoSONE12(2):e0173008.doi:10.1371/journal. murinemodelsofinfluenzavirusinfectioninasthmaticsshowthatasthmaticmicearehighly
pone.0173008
resistanttoinfluenzavirusinfection,whichcontradictsclinicalobservationsinhumans.
Editor:BernhardRyffel,CentreNationaldela Here,wedevelopedamurinemodelofinfluenzavirus/asthmacomorbidityusingNC/Nga
RechercheScientifique,FRANCE
mice,whicharehighlysensitivetoallergicreactionssuchasatopicdermatitisandallergic
Received:December8,2016 airwayinflammation.Thismodelwasthenusedtoexaminetheimpactofallergicairway
Accepted:February13,2017 inflammationonlungpathologyinthe2009pandemicinfluenzavirusinfectedmice.The
resultsshowedthatinductionofacuteallergicairwayinflammationinpre-existinginfluenza
Published:February28,2017
virusinfectionhadadditiveeffectsonexacerbationoflungpathology,whichmirrorsfindings
Copyright:©2017Kawaguchietal.Thisisanopen
inhumanepidemiologicalstudies.Incontrast,pre-existingallergicairwayinflammationpro-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which tectedfromsubsequentinfluenzavirusinfection,whichwascompatiblewiththoseofprevi-
permitsunrestricteduse,distribution,and ousmurinemodelsofinfluenzavirusinfectioninasthmaticmice.Thesevariableoutcomes
reproductioninanymedium,providedtheoriginal
ofthismurinemodelindicatethatthetemporalrelationbetweenallergicairwayinflammation
authorandsourcearecredited.
andinfluenzavirusinfectionmightplayacriticalroleinasthmaandinfluenzacomorbidity.
DataAvailabilityStatement:Allrelevantdataare
Thus,thismurinemodelwillfurtherourunderstandingofhowinfluenzavirusinfection
withinthepaper.
affectsanasthmatichostandmayaidthedevelopmentofstrategiestoimprovetreatments
Funding:Theworkdescribedinthisreportwas
andoutcomesforasthmaticsharboringinfluenzavirusinfection.
supportedbygrantsfromtheJapaneseMinistryof
Health,Labor,andWelfare,andResearchProgram
onEmergingandRe-emergingInfectiousDiseases
fromJapanAgencyforMedicalResearchand
Development.Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
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Anovelcomorbiditymousemodelofasthmaandinfluenza
Competinginterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
InfluenzaAvirusistheetiologicalagentofinfluenza,whichischaracterizedbysuddenonsetof
highfeverandrespiratorysymptomssuchascoughandsorethroat,aswellasbysystemicsymp-
tomssuchasheadache,muscleaches,andfatigue.Influenzavirusescontinuallycirculatein
humanpopulationsworldwide.Annualepidemicsresultinapproximatelythreetofivemillion
casesofsevereillnessandapproximately250,000to500,000deaths[1].Theinfluenzapandemic
of2009wascausedbyanoveltriplereassortantinfluenzavirus,A(H1N1)pdm2009,whichfirst
emergedinpigsinMexicobutspreadrapidlyacrosstheglobe[2].Thepandemicinfluenzaocca-
sionallycausedseverelowerairwayinflammationandpneumonia.Amongthepatientswiththe
2009pandemicinfluenzavirusinfection,itwasnotedthatasthmawasthemostcommon
comorbidityamongpatientshospitalizedwithinfluenzaduringthispandemic[3][4].
Asthmaisachronicinflammatoryairwaydisorderaffectingtheentirebrachialtreeanddis-
tressing300millionspeopleworldwide,makingitamajorpublichealthconcern[5].Inhalation
ofspecificallergensorotherenvironmentalmaterialsinasthmainducesacuteairwayhypersensi-
tivity,smoothmusclecontraction,mucosaledema,andmucushypersecretionresultinginairway
obstruction,whichcausessymptomsincludingcoughing,wheezing,andchesttightness[6][7].
Airwayobstructionisvariable,withsymptom-freeperiodsinterspersedbyperiodsofrepeated
exacerbations[8].Respiratoryvirusinfectionsarethemostcommoncauseofasthmaexacerba-
tion[9].Thevirusmostcommonlyassociatedwithasthmaexacerbationisrhinovirus,followed
bycoronavirus,respiratorysyncytialvirus,parainfluenzavirus,andinfluenzavirus[10].
Animalmodelsofhumaninfectiousdiseaseshavecrucialrolesinelucidationofthepatho-
genesis,developmentofpreventionsandtreatmentsoftheinfectiousdiseases.Similarity
betweenhumandiseasephenotypesandanimalmodelphenotypesiscriticalfactorforelucida-
tionofthepathogenesisofthehumandiseases.However,susceptibilitiesofanimalstohuman
pathogensdiffersignificantlydependingonanimalspeciesandstrains.Then,phenotypesof
infectiousdiseasesonanimalmodelmaydifferslightlyfromthehumandiseasesrequiring
carefulinterpretationoftheanimalmodelphenotypes.Existingmurinemodelsofasthmawith
respiratoryvirusinfectionhaveshowninfluenzavirusesareinvolvedinasthmadevelopment
[11].However,othermurinemodelsdevelopedtoexaminethepathogenesisofinfluenza/aller-
gicasthmashowthatasthmaticmicearehighlyresistanttoinfluenzavirusinfection[12–14],
whichcontradictsthefindingsofepidemiologicalstudiesinhumans,implyingthatfurther
studiesusingotheranimalmodelsareneededtoelucidatethepathogenesisofthiscomplicated
comorbidityofinfluenzavirusinfectioninasthmatics.Anovalbumin(OVA)-sensitizedand
OVA-challengedmurinemodel(basedonBALB/cmice)isoneofthemostpopularand
provenmodelsofallergicairwayinflammation[15].However,itisoftendifficulttoreproduce
complicatedclinicalsettingsfaithfullywithanimalmodelsusingonlyafewinbredlaboratory
micestrainsbecausedifferentlaboratorystrainsofmiceshowdifferentsusceptibilitiesand
responsestoantigens[16,17].Aninbredstrainoflaboratorymice,NC/Nga,hasbeenusedas
ananimalmodelofhumanatopicdermatitis[18–20]becausetheyharbormutationsonchro-
mosome9thatarelinkedtoincreasedIgEproductionandincreasedTh2responses[21].
Repeatedadministrationofallergenstotheskinofthesemiceinducesatopicdermatitis-like
skinlesions;thisdoesnothappeninBALB/cmice[22,23].Moreover,massiveandprolonged
allergicresponses,suchaseosinophilicinfiltrationandIgEproductionwithsevereairway
inflammation,wereobservedinOVA-sensitizedandOVA-challengedNC/Ngamice,butnot
inBALB/cmice,indicatingthatNC/Ngamicearemoresensitivetoseveraltypesofallergic
disease/reaction(includingallergicairwayinflammation)thanBALB/cmice[24,25].
Here,wedevelopedaninfluenzavirus-infection/atopicmousecomorbiditymodelbasedon
NC/Ngamiceanda2009pandemicinfluenzavirus,A/Narita/1/2009(H1N1)pdm09.Usingthe
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Anovelcomorbiditymousemodelofasthmaandinfluenza
mousemodel,wehaveinvestigatedtheimpactofallergenchallengesonlungpathologyinthe
pandemicinfluenzavirusinfectedmice.Weobservedanacuteallergicinflammationintheair-
waysofasthmaticmicesoonafterinfluenzavirusinfectionenhancedofpulmonaryinflamma-
tion,subsequentepithelialgobletcellmetaplasiaandweightlosswithoutaffectingvirus
replication,raisingahypothesisthattheallergenchallengesontopofinfluenzavirusinfection
inducedinflammationhadadditiveeffectsonexacerbationoflungtissuedamage.Thefinding
wascompatiblewiththoseofprevioushumanclinicalobservations.Incontrast,pre-existing
allergicairwayinflammationinhibitedsubsequentinfluenzavirusinfection,whichwasconsis-
tentwiththepreviousmurinemodelsofinfluenza/allergicasthma.Thesetwodifferentoutcomes
ofthismurinemodelsuggestthatthetemporalrelationbetweeninfluenzavirusinfectionand
allergicairwayinflammationmightbeoneofthedeterminantsforcomorbidityofinfluenza
virusinfectioninasthmatics.Thus,thismurinemodelwillcontributetoourunderstandingof
howinfluenzavirusexacerbatesandenhancesairwaypathologyinanasthmatichost.
Materialsandmethods
Animals
NC/Ngamice(female,6to8weeksold)bredunderspecificpathogen-freeconditionswere
purchasedfromJapanSLCInc.,Shizuoka,Japan.Animalstudieswereperformedinstrict
accordancewiththeGuidelinesforProperConductofAnimalExperimentsoftheScientific
CouncilofJapan.Allanimalexperimentswereconductedinstrictcompliancewithanimal
husbandryandwelfareregulationsinhandledinbiosafetyleveltwoanimalfacilitiesaccording
totheguidelinesoftheAnimalCareandUseCommitteeoftheNationalInstituteofInfectious
Diseases,andwereapprovedbythisCommittee(approvalnos.110032,112133,114064,
211055,and213069).Miceweremonitoreddailyforclinicalsignsofmorbidityandmortality
upto21dayspostinfection.Thehumanendpointwasusedformicethatlost25%ormoreof
theirinitialbodyweightduringthestudy.Whentheanimalsmetthecriteria,theywerescored
deadandeuthanizedunderexcessisofluraneanesthesiaaccordingtoinstitutionalguidelines.
Virus
Mouse-adaptedinfluenzavirusA/Narita/1/2009,whichwasobtainedafter15passagesin
mice,wasusedtorepresenttheA(H1N1)pdm09virus(pH1N1)inthisstudy[26].Thevirus
wasgrownintheallantoiccavitiesof10-to-11-day-oldfertilechickeneggs.Micewereinfected
viaintranasal(IN)administrationof20μlofPBScontainingpH1N1virussuspension(40,000
plaque-formingunits(PFUs)/mouse)intotheleftnostril.
Measurementofvirustiter
ThevirustiterwasmeasuredaccordingtothemethodofTobitaetal.[27].Briefly,200μlali-
quotsofbronchoalveolarlavagefluid(BALF,seriallydiluted10-fold)wereinoculatedinto
MDCKcellsinasix-wellplate.After1h,eachwellwaswashedtoremovenon-attachedvirus,
andthenoverlaidwith2mlofagarmedium.Thenumberofplaqueswascountedbycrystal
violetstainingat2dayspost-inoculation.Thevirustiterwasexpressedaspfu/ml.
Mousemodelsofallergicairwayinflammation
ANC/NgamousemodelofOVA-inducedallergicairwayinflammationwasusedinthisstudy,
butwithslightmodification[25,28,29].SixtoeightweekoldfemaleNC/Ngamiceweresensi-
tizedbytwoconsecutiveweeklyintraperitoneal(IP)injectionsof100μgofOVA(Sigma-
Aldrich,St.Louis,MO)plus2mgofalumcrystals(InvivoGen,SanDiego,CA).Oneweek
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Anovelcomorbiditymousemodelofasthmaandinfluenza
afterthesecondIPsensitization,themicewereanesthetizedbyintraperitonealinjectionofa
mixtureof1.0mgketamineand0.02mgxylazinein0.1ml/10gbodyweightandsubjectedto
INchallengewith20μlofpH1N1virus.Control(Mock,non-infected)micewereinoculated
withdilutedallantoicfluidlackingvirus.Onthefollowing2days,thevirus-infectedmicewere
thenanesthetizedbyintraperitonealinjectionofamixtureof1.0mgketamineand0.02mg
xylazinein0.1ml/10gbodyweightandchallengedINwith100μgofOVAin20μlofPBS.
ControlmicethatdidnothaveallergicairwayinflammationwerechallengedwithPBSinstead
ofOVA.Non-infectedmice(10micepergroup)andpH1N1-infectedmice(20miceper
group)werethenmonitoredforweightlossuntil14daysafterviralinfection.Themicewere
sacrificedunderexcessisofluraneanesthesiaon2,3,or21dayspost-infectionandsubjectedto
collectionofbronchoalveolarlavagefluid(n=5micepergroupon2and3dayspost-infec-
tion)andpathologicalanalysis(n=3micepergroupon3dayspost-infection,n=5–11mice
pergroupon21dayspost-infection).Totesttheeffectofpre-existingacuteallergicairway
inflammation,1weekafterthesecondIPsensitizationwithOVA,themicewereanesthetized
byintraperitonealinjectionofamixtureof1.0mgketamineand0.02mgxylazinein0.1ml/10
gbodyweightandchallengedINwith100μgofOVAin20μlofPBSfor2consecutivedays.
ControlmicethatdidnothaveallergicairwayinflammationwerechallengedwithPBSinstead
ofOVA.Then,onthenextdayafterthesecondINchallengewithOVA,themicewereanes-
thetizedbyintraperitonealinjectionofamixtureof1.0mgketamineand0.02mgxylazinein
0.1ml/10gbodyweightandchallengedINwith20μlofpH1N1virus.Control(Mock,non-
infected)micewereinoculatedwithdilutedallantoicfluidlackingvirus.Miceinfectedwith
pH1N1(n=10micepergroup)werethenmonitoredforweightlossuntil15daysafterviral
infection.BodyweightonDay0(immediatelybeforevirusinfection)wasconsideredtobe
100%.Miceweresacrificedunderexcessisofluraneanesthesiaon0(immediatelybeforevirus
infection),3,or21dayspost-infectionandsubjectedtocollectionofbronchoalveolarlavage
fluid(n=5micepergroupon0and3dayspost-infection)andpathologicalanalysis(n=3
micepergroupon3dayspost-infection,n=10micepergroupon21dayspost-infection).For
theexperimentsinvolvingneuraminidaseinhibitortreatment,1weekafterthesecondIPsen-
sitizationwithOVAthemicewereanesthetizedbyintraperitonealinjectionofamixtureof1.0
mgketamineand0.02mgxylazinein0.1ml/10gbodyweightandchallengedINwith20μlof
pH1N1virus.Onthefollowing2days,thevirus-infectedmicewereanesthetizedbyintraperi-
tonealinjectionofamixtureof1.0mgketamineand0.02mgxylazinein0.1ml/10gbody
weightandchallengedINwith100μgofOVAwithorwithout800μgofzanamivir(ZAN)in
50μlofPBS.Themiceweresacrificedunderexcessisofluraneanesthesiaon3or21days
(pH1N1-infectedmice)afterinfectionandsubjectedtocollectionofbronchoalveolarlavage
fluid(n=5micepergroupon3dayspost-infection)andpathologicalanalysis(n=3miceper
groupon3dayspost-infection,n=5–10micepergroupon21dayspost-infection).Control
(Mock,non-infected)micewereinoculatedwithdilutedallantoicfluidlackingvirus.Another
groupofcontrolmicethatdidnothaveallergicairwayinflammationwerechallengedwith
PBSinsteadofOVA.
Collectionofbronchoalveolarlavagefluid(BALF)
BALFwasharvestedbydelivering1mlofPBStothelungsthroughatrachealcannula.The
numberandtypeofcells,virustiter,andlevelsofcytokinesandchemokinesintheBALFwere
thenmeasured.ThenumberofcellsintheBALFwascountedusingVetscanHM5(Abaxis
VeterinaryDiagnostics,UnionCity,CA).Toconfirmthemorphologyofthecells,50μlof
BALFwascentrifugedontoglassslidesat1000rpmfor10minusingaShandoncytocentrifuge
(ThermoFisherScientificInc.,Waltham,MA).ThecellswerethenstainedwithGiemsaand
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Anovelcomorbiditymousemodelofasthmaandinfluenza
analyzedbymicroscopy.TherestoftheBALFwascentrifugedfor5minat500×gat4˚Cto
removecelldebrisandstoredat−80˚Cuntilrequired.
AnalysisofcytokinesandchemokinesinBALF
Thelevelsofinflammatorycytokines/chemokinesintheBALFweremeasuredusingthe
MouseCytokineTwenty-PlexAntibodyBeadKit(Invitrogen,SanDiego,CA)with2-fold
dilutedBALFasananalyte,asdescribedbythemanufacturer.Theseassayscandeterminethe
concentrationsofthefollowing20cytokinesandchemokines:granulocyte-macrophagecol-
ony-stimulatingfactor(GM-CSF),gammainterferon(IFN-γ),interleukin1α(IL-1α),IL-1β,
IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,IL-13,IL-17,gammainterferon-inducedprotein10(IP-10/
CXCL10),fibroblastgrowthfactor-basic(FGF-Basic),neutrophil-relatedchemokineKC(KC/
GRO),monocytechemoattractantprotein1(MCP-1/CCL2),macrophageinflammatorypro-
tein1α(MIP-1α/CCL3),monokineinducedbygammainterferon(MIG/CXCL9),tumor
necrosisfactoralpha(TNF-α),andvascularendothelialgrowthfactor(VEGF).TypeIIFNsin
BALFweremeasuredusingmousealphaandbetaIFN(IFN-α,and-β)enzyme-linkedimmu-
nosorbentassay(ELISA)kits(PBLInterferonSource,Piscataway,NJ),accordingtotheproto-
coldescribedbythemanufacturer.Forexperimentsinvolvingneuraminidaseinhibitor
treatment,themouseCCL2/JE/MCP-1DuoSetELISA(R&DSystemsInc.,Minneapolis,MN)
wasusedtomeasuretheconcentrationofMCP-1/CCL2.
Histopathologyandimmunohistochemistry
Animalsweresacrificedat3daysor21dayspost-infection.Excisedlungtissueswerefixedin
10%phosphate-bufferedformalin,whichwasinjectedintothetracheauntilthelungsinflated.
Allthelobesofthefixedlungtissueswerethenembeddedinparaffin,sectioned,andstained
withhematoxylinandeosin(H&E)orPeriodicacid-Schiff(PAS).ThenumberofPASpositive
epithelialcellsinlungtissueswasobtainedbyvisualestimationovertheentireareaofthesec-
tion.ImmunohistochemistrywasperformedwitharabbitpolyclonalantibodyfortypeA
influenzanucleoprotein(IAVNP)antigen(preparedinourlaboratory[30])astheprimary
antibody,aspreviouslydescribed[31].Aperoxidase-labeledpolymer-conjugatedanti-rabbit
immunoglobulin(EnVision/HRP,Dako,Glostrup,Denmark)wasusedasthesecondaryanti-
body.Peroxidaseactivitywasdetectedbydiaminobenzidine/hydrogenperoxide.
Statisticalanalysis
StatisticalanalysiswasperformedusingtheGraphPadPrismstatisticalsoftwarepackage(Ver-
sion5.0c:GraphPadSoftwareInc.,CA).Comparisonsbetweentwogroupswereperformed
usinganunpairedstudent’st-testorMann-WhitneyUtest.Comparisonsbetweenmorethan
threegroupswereperformedusingone-wayANOVAfollowedbyTukey’smultiplecompari-
sonstestornon-parametricone-wayANOVA(Kruskal-Wallistest)followedbyDunn’smulti-
plecomparisontests.Analyzingdatawithtwoindependentvariables,suchasbodyweight
measurements,wasperformedusingtwo-wayANOVAfollowedbySidak’smultiplecompari-
sonstest.Datawereconsideredstatisticallysignificantatp<0.05.
Results
Acuteallergicairwayinflammationexacerbateslungpathologyin
influenzavirusinfectedmicewithoutaffectingpre-existingvirusinfection
Thefirstmurinemodelofallergicairwayinflammationwassetuptoexaminetheeffectofa
pre-existinginfluenzavirusinfectiononsubsequentallergicairwayinflammation.NC/Nga
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Anovelcomorbiditymousemodelofasthmaandinfluenza
miceweresensitizedtoOVA(amodelallergen)byrepeatedIPinjectionwithaluminum
hydroxideastheadjuvant.Thesensitizedmiceweretheninfectedwithasublethaldoseof
influenzavirusA/H1N1pdm09(pH1N1).Subsequently,theinfectedmicewerechallengedIN
withOVAfortwoconsecutivedays(Fig1A).ThepH1N1-infected/OVA-challengedmice
(pH1N1/OVA)exhibitedweightlossmuchsevererthanmicenotsubjectedtoOVAchallenge
(pH1N1/PBS);however,non-infectedmicewithandwithoutOVAchallenge(Mock/OVA
andMock/PBS,respectively)didnotloseweight(Fig1B).However,theviraltiterinBALF
frompH1N1-infectedmicewasnotaffectedbyOVAchallenge(Fig1C).Bothallergenchal-
lengesandrespiratoryinfectionsinducepulmonaryinflammation,althoughdifferenttypesof
immunecellstakeprecedenceduringthesereactions.Histopathologicalanalysisrevealedthat
thelungtissuesofpH1N1/OVAmiceshowedmassiveinfiltrationofinflammatorycells,
includingeosinophils(Fig1Dleftpanelinset),tothebronchiolesandalveoliatDay3post-
infection(Fig1Dleftpanel).Bycontrast,thelungsofpH1N1/PBSmiceshowedlimited
inflammationofperivascularalveolarareas(Fig1D,leftmiddlepanel).ThelungsofMock/
OVAmicealsoshowedmildeosinophilicinflammationaroundbronchioleswhilethoseof
Mock/PBSmiceremainedintact,confirmingthatacuteallergicairwayinflammationwas
inducedbythechallengesofOVAbyINroutefortwoconsecutivedaysinthesemice(Fig1D,
rightmiddleandrightpanels).Significantincreasesintotalcellnumbersandtheproportion
ofeosinophils,whichareinvolvedinacuteallergicairwayinflammation,wereobservedinthe
BALFfrompH1N1/OVAmiceatDay3post-infectionwhencomparedwiththoseinthe
BALFfrompH1N1/PBSmice(Fig1E),andalsoobservedintheBALFfromMock/OVAmice
whencomparedwiththoseintheBALFfromMock/PBSmice(Fig1F)Bycontrast,thediffer-
encebetweentheproportionsofneutrophils,whichistriggeredbyinfluenzavirusinfection,in
thesemicewasnotsignificant(Fig1E).Takentogether,theseobservationsindicatethatOVA-
inducedacuteallergicairwayinflammationuponinfluenzavirusinfectionsynergisticallyexac-
erbatethelungpathologywithoutaffectinginfluenzavirusreplicationorvirus-inducedneu-
trophilicinflammation.
Influenzavirusinfectionandsubsequentacuteallergicairwayresponse
synergisticallyenhancechemokineproduction
Ascytokinesandchemokinesinducedbyvirusinfectionorallergicresponsearecrucialmole-
culesthatregulateinflammationprocessesinthelung,andbecausetheirconcentrationscorre-
latewiththeseverityofillness,wemeasuredtheamountsofvariouscytokinesandchemokines
intheBALFofmice.WefoundthatexpressionsofMCP-1/CCL2,MIP-1α/CCL3,andIP-10/
CXCL10weresignificantlyhigherinpH1N1/PBSandMock/OVAmicethaninMock/PBS
mice.Furthermore,thelevelsofthesethreechemokinesinpH1N1/OVAmiceweremarkedly
higherthanthoseintheotherthreegroups(Fig2A).Incontrast,thelevelsofIL-4,IL-6,IL-12,
TNF-α,GM-CSF,andVEGF(Fig2B),andthoseofIFN-α,IFN-β,andIFN-γ(Fig2C),in
pH1N1/OVAmicewerenothigherthanthoseinpH1N1/mockmicewhilethelevelsofthese
cytokinesandinterferonsinpH1N1/PBSmicewerehigherthanthoseinMock/PBSmice(Fig
2Band2C).Theseobservationssuggestedthatacuteallergicimmuneresponsesintheairways
soonafterinfluenzavirusinfectionhadsynergisticeffectsofproductionsofMCP-1/CCL2,
MIP-1α/CCL3,andIP-10/CXCL10withinfluenzavirusinfectioninducedimmuneresponses
andthatthesechemokinesmightplayapivotalroleinexacerbatingthepulmonaryinflamma-
tionininfluenzavirusinfectedmicewithsubsequentallergenchallenges.Bycontrast,although
thelevelsofIL-5andIL-13weresignificantlyhigherinMock/OVAmicethaninMock/PBS
mice,theyweremarkedlylowerinpH1N1/OVAmicethaninMock/OVAmice(Fig2D),
confirmingthattheexacerbationofthelungpathologyobservedinthismodelmightbe
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Fig1.InfluenzavirusinfectionandsubsequentinductionofacuteallergicairwayresponsesbyOVAinOVA-sensitizedNC/
Ngamice.(A)SchematicrepresentationoftheprotocolforOVAsensitization,influenzavirusinfection,andOVA-inducedallergic
airwayresponses.NC/NgamiceweresensitizedtwicebyIPinjectionofOVAonDay-14andDay-7andtheninfectedwitha
sublethaldoseofinfluenzavirusA/H1N1pdm09(pH1N1)orMockallantoicfluid(Day0).Onthe2daysfollowinginfection,themice
werechallengedagainwithOVAorPBSviatheINroute(Day+1andDay+2).(B)Weightlosswasmonitoredafterinfection(20mice
inthepH1N1infectiongroupsand10miceintheMockgroups).Errorbarsrepresentthemean±SEM.Weightchangedatawere
combinedfromthree(pH1N1infectiongroups)ortwo(Mockgroups)independentexperiments.(C)OnDay3post-infection,virus
titersintheBALFweremeasuredinaplaqueassay(5mice/group).Dataareexpressedasscatterplotswiththemeanviraltiter±SD.
(D)RepresentativelungtissuesectionsfrompH1N1/OVA,pH1N1/PBS,Mock/OVA,andMock/PBSmiceonDay3post-infection(3
mice/group).Sectionswerestainedwithhematoxylinandeosin(H&E).Originalmagnification:×10or×40(inset).(E)Totalcell
countsandtheproportionofinflammatorycellsintheBALFformpH1N1/PBSandpH1N1/OVAmiceassessedonDay2andDay3
post-infection(5mice/group).Dataareexpressedasscatterplotswiththemean±SD.(F)Totalcellcountsandtheproportionof
inflammatorycellsintheBALFformMock/PBSandMock/OVAmiceassessedonDay2andDay3post-infection(5mice/group).
Dataareexpressedasscatterplotswiththemean±SD.*p<0.05,**p<0.01,***p<0.001,and****p<0.0001(two-wayANOVAor
Mann-WhitneyUtest).IP,intraperitoneal;IN,intranasal;BALF,bronchoalveolarlavagefluid;ns,notsignificant;Br,Bronchiole.The
experimentswererepeatedindependentlyatleasttwice.
doi:10.1371/journal.pone.0173008.g001
attributabletosynergisticincreaseofchemokinesproductioninducedbybothofpre-existing
influenzavirusinfectionandsubsequentallergicairwayinflammationandnottodeterioration
ofacuteallergicairwayinflammationwhichisaTh2-driveninflammatoryresponse.
Pre-existingacuteallergicairwayinflammationprotectsfrom
subsequentinfluenzavirusinfectionandreducesvirus-inducedcytokine
andchemokineproduction
Recentreportsshowedthatacuteallergicairwayinflammationacceleratestheclearanceof
influenzavirus[12–14],whichappearstocontradictourpresentobservations.Toexplorethe
reasonsfortheseapparentdifferences,wemodifiedtheexperimentalprotocolbyreversingthe
orderoftheinterventions.NC/NgamicewerefirstsensitizedwithOVAbyrepeatedIPinjec-
tionofOVAwithaluminumhydroxideastheadjuvant.Then,thesensitizedmicewerechal-
lengedINwithOVAfortwoconsecutivedays.OnthedayofthelastOVAchallenge,themice
wereinfectedwithasublethaldoseofpH1N1(Fig3A).Histopathologicalanalysisofthelungs
fromthepH1N1-infectedmicethatwerepre-challengedwithOVA(OVA/pH1N1)revealed
thatinflammationwaslimitedandsimilartothatinthelungsfromthepH1N1-infectedmice
thatwerenotpre-challengedwithOVApriortoviralinfection(PBS/pH1N1)atDay3post-
infection(Fig3Bleftandleftmiddlepanels).Thelungsofnon-infectedmicewithOVAchal-
lenge(OVA/Mock)showedsubtleeosinophilicinfiltrationsaroundbronchioles,confirming
thattheallergenchallengesinthisprotocolinducedacuteallergicairwayinflammation(Fig
3B,rightmiddlepanel).Interestingly,OVA/pH1N1micedidnotshowweightlosswhilePBS/
pH1N1micesufferedsignificantweightloss(Fig3C).Wealsoobservedsignificantreductions
intheviraltiterintheBALFfromOVA/pH1N1mice(Fig3D).Theproportionsofeosino-
phils,whichareinvolvedinacuteallergicairwayinflammation,werehigherinBALFfrom
micewithOVAchallenge(OVA/MockorOVA/pH1N1)thaninthosefrommicewithout
OVAchallenge(PBS/MockorPBS/pH1N1)regardlessofinfluenzavirusinfectionbefore(Day
0)andafter(Day3)infection.Inaddition,totalcellnumbersintheBALFfrommicewith
OVAchallengewerehigherthanthoseintheBALFfrommicewithoutOVAchallengebefore
infection(Day0),whereasthedifferencesbetweenthenumbersinthesemicewerenotsignifi-
cantregardlessofinfluenzavirusinfectionatDay3(Fig3E).Asignificantincreaseinthepro-
portionofneutrophils,whichistriggeredbyinfluenzavirusinfection,wasobservedinthe
BALFfrombothOVA/pH1N1miceorPBS/pH1N1miceatDay3post-infectionwhencom-
paredwiththoseintheBALFfrommicewithoutinfection(OVA/MockorPBS/Mock,respec-
tively)regardlessofOVAchallenge(p<0.001,OVA/pH1N1vs.OVA/Mock;p<0.001,PBS/
PLOSONE|DOI:10.1371/journal.pone.0173008 February28,2017 8/22
Anovelcomorbiditymousemodelofasthmaandinfluenza
Fig2.ConcentrationsofchemokinesandcytokinesinBALFfrommiceafterinfluenzavirusinfectionandsubsequentinductionof
allergicairwayresponses.TheconcentrationsofchemokinesandcytokinesinBALFfrompH1N1/OVA,pH1N1/PBS,Mock/OVA,and
PLOSONE|DOI:10.1371/journal.pone.0173008 February28,2017 9/22
Anovelcomorbiditymousemodelofasthmaandinfluenza
Mock/PBSmiceweremeasuredonDay2and3post-infection(5mice/group).(A)ConcentrationsofMCP-1/CCL2,MIP-1α/CCL3,andIP-10/
CXCL10inBALF.(B)Concentrationsofcytokines,IL-4,IL-6,IL-12,TNF-α,GM-CSF,andVEGF,inBALF.(C)ConcentrationsofIFN-α,IFN-
β,andIFN-γinBALF.(D)ConcentrationsofIL-5andIL-13inBALF.Dataareexpressedasscatterplotswiththemean.*p<0.05,**p<0.01,
***p<0.001,and****p<0.0001(two-wayANOVA).Theexperimentswererepeatedtwiceindependently.
doi:10.1371/journal.pone.0173008.g002
pH1N1vs.PBS/Mock).However,thedifferencebetweentheproportionsofneutrophilsin
OVA/pH1N1andPBS/pH1N1micewasnotsignificantatDay3post-infection(Fig3E).
Takentogether,theseobservationsindicatedthatpre-existingOVA-inducedacuteallergicair-
wayinflammationinhibitedinfluenzavirusreplicationwithoutaffectingvirus-inducedinflam-
matorycellsresponsesintheearlyphaseofinfection.Then,wemeasuredtheamountsof
proinflammatorycytokinesandchemokinesintheBALFofmice.Beforeinfection(Day0),
MCP-1/CCL2,MIP-1α/CCL3,andIP-10/CXCL10levelswerehigherinOVAchallengedmice
(OVA)thanthoseinmicewithoutOVAchallenge(PBS)(Fig3F),whereasthelevelsofthese
chemokinesinOVA/pH1N1micefellbyDay3post-virusinfectionandthelevelsinPBS/
pH1N1werehigherthanthoseinOVA/pH1N1mice(Fig3F).Inaddition,thesechemokine
levelsinmicewithoutvirusinfectionatDay3werediminished.AtDay3post-infection,expres-
sionofIL-6,IL-12,GM-CSF,IFN-α,IFN-β,andIFN-γwasalsosignificantlyhigherinmicenot
pre-challengedwithOVA(PBS/pH1N1)thaninmicethatwerepre-challenged(Fig3Gand
3H).Meanwhile,thelevelsofIL-5andIL-13,whichareTh2cytokines,weresignificantlyhigher
intheBALFofOVAmicethanthoseofPBSmicebeforeinfection(Day0)althoughthey
declinedafterinfection(Day3)regardlessofvirusinfection(Fig3I).Takentogether,these
resultssuggestedthatapre-existingacuteallergicimmuneresponseinhibitedthereplicationof
influenzavirus,decreasedvirus-inducedcytokineandchemokineproduction,andreducedthe
severityofinfluenzavirus-inducedillness,whichiscompatiblewithpreviousfindings[12–14].
Pre-existingacuteallergicairwayinflammationsuppressesinfluenza
virusinfectionofthebronchiolarepithelium
Thetwomurinemodelsdescribedabove(i.e.,theeffectofallergicairwayresponsesonpre-
existinginfluenzavirusinfection(Figs1and2)andtheeffectofinfluenzavirusinfectionon
pre-existingacuteallergicairwayinflammation(Fig3)),showconflictingresultswithrespect
totherelationshipbetweenacuteallergicairwayinflammationandinfluenzavirusinfection.
Toaddressthereasonsforthisdiscrepancy,weperformedimmunohistochemicalanalysisof
lungtissuesusingananti-influenzaNPantibodytoexaminethedistributionofviralinfected
cells.Wedetectedsimilarlevelsofinfluenzavirusantigensinboththebronchiolarandalveolar
epitheliumofpH1N1/OVAandpH1N1/PBSmiceatDay3post-infection,irrespectiveofsub-
sequentOVAchallenge(Fig4A).Bycontrast,comparedtomicenotpre-challengedwith
OVA(PBS/pH1N1),weobservedamarkedreductionintheamountofinfluenzavirusantigen
inthebronchiolarepitheliumofmicethatwerepre-challengedwithOVA(OVA/pH1N1),
despitethepresenceofabundantinfectedcellsinthealveolarepithelium(Fig4B).These
observationsindicatedthatpre-existingacuteallergicairwayinflammationsuppressedthe
influenzavirusreplicationspecificallyinthebronchiolarairwayepithelium(thesiteaffected
byasthmaandallergicairwayinflammation)butnotinthealveolarepithelium(whichisunaf-
fectedbyasthmaandallergicairwayinflammation);thuspre-existingacuteallergicairway
inflammationdecreasedthelevelofvirus-inducedcytokineandchemokineproduction,and
thenreducedtheseverityofinfluenzavirus-inducedillness.Ontheotherhand,subsequent
allergicairwayinflammationininfluenzavirusinfectedlungappearstohaveanegligibleeffect
onviralreplicationinthebronchiolarepithelium,whichtriggersexcessivechemokineproduc-
tionandexacerbateslungpathology.
PLOSONE|DOI:10.1371/journal.pone.0173008 February28,2017 10/22